Identification of a novel ipp gene cluster responsible for 2-isopropylphenol degradation in strain Rhodococcus sp. D-6.

Isoprocarb (IPC), a widely used carbamate insecticide, causes environmental contamination and poses risks to humans and ecosystems. Rhodococcus sp. D-6, capable of utilizing IPC as the sole growth substrate, was isolated by our lab. Strain D-6 initiates IPC degradation through ester bond hydrolysis by the hydrolase IpcH, yielding 2-isopropylphenol (IPP). In this study, the catabolic pathway of IPP in strain D-6 was elucidated, and the ipp gene cluster responsible for its degradation was predicted by transcriptomic analysis. Key genes, including a novel two-component IPP monooxygenase gene ippA1A2 and a 2-isopropylhydroquinone dioxygenase gene ippB, are responsible for the successive conversion of IPP, resulting in the ring cleavage of IPP. IppA1, a group D flavin-dependent monooxygenase, along with its reductase component IppA2, hydroxylates IPP to 2-isopropylhydroquinone using reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) as cofactors. The optimal molar ratio of IppA1 to IppA2 was 3:1. The apparent K_m and k_cat values of IppA1 for IPP were 60.52 ± 1.27 µM and 70.07 ± 0.85 s^-1, respectively. IppB, an extradiol dioxygenase, catalyzes 2-isopropylhydroquinone ring cleavage to generate 2-isopropyl-4-hydroxymuconic semialdehyde. The apparent K_m and k_cat values of IppB for 2-isopropylhydroquinone were 29.07 ± 1.54 μM and 147.65 ± 1.92 s^-1, respectively. This study provides critical insights into the molecular mechanisms of IPP degradation, contributing to a comprehensive understanding of IPC degradation.IMPORTANCECarbamate insecticides kill pests by inhibiting the activity of acetylcholinesterase (AChE) and have been widely used in agriculture. Compared to the studies on the degradation mechanisms of carbofuran and carbaryl, little is known about IPC degradation. An IPC-degrading strain Rhodococcus sp. D-6 was isolated by our lab, and the hydrolase gene ipcH responsible for hydrolyzing IPC to IPP has been identified previously. This study further elucidates the IPP degradation pathway in strain D-6 and identifies the novel two-component IPP monooxygenase IppA1A2 and 2-isopropylhydroquinone dioxygenase IppB, which are respectively responsible for IPP hydroxylation and its subsequent ring cleavage. These findings enhance our understanding of the microbial degradation mechanism of IPC.